Several methods have been described for the synthesis of oligonucleotides using phosphoramidite chemistry, which are now capable of achieving nucleotide coupling efficiencies of 99%. The primary markets for commercial oligonucleotide synthesis are synthesis of oligonucleotide arrays for genomic and expression applications, and for use as PCR primers, for which such efficiencies are adequate. Since 1990 there has been little work done improving oligonucleotide chemistries to increase coupling efficiencies, the focus has instead been on increasing throughput with existing chemistries. Increased coupling efficiencies would provide a significant benefit to growing applications such as synthesis of long polynucleotides by assembly of oligonucleotides, accurate detection of single nucleotide polymorphisms in individuals and populations, the manufacture of high quality microarray chips for use in clinical diagnostics, haplotyping, real-time polymerase chain reaction, small inhibitory RNAs (siRNAs) used for validation of drug targets, expression array production, and chip-based sequencing. There is therefore a need in the art for synthetic processes that reduce synthesis errors and increase oligonucleotide coupling efficiencies.
Several methods have been described for the synthesis of larger polynucleotides by the assembly of oligonucleotides, using combinations of ligation, polymerase chain reaction and ligase chain reaction. See, for example, Hayden et al., 1988, DNA 7, 571-7; Ciccarelli et al., 1991, Nucleic Acids Res 19, 6007-13; Jayaraman et al., 1991, Proc Natl Acad Sci USA 88, 4084-8; Jayaraman et al., 1992, Biotechniques 12: 392-8; Graham et al., 1993, Nucleic Acids Res 21: 4923-8; Kobayashi et al., 1997, Biotechniques 23: 500-3; Au et al., 1998, Biochem Biophys Res Commun. 248: 200-203; Hoover et al., 2002, Nucleic Acids Res 30: e43, each of which is hereby incorporated by reference in its entirety. The assembly of polynucleotides from oligonucleotides is an error-prone process. Errors arise from the chemical synthesis of oligonucleotides, and the enzymatic processes used to assemble these oligonucleotides into longer polynucleotides. These errors increase the cost and time taken to synthesize polynucleotides. There is therefore a need in the art for synthetic processes that reduce synthesis errors and synthesis time.